Compositions and methods for treating cancer using immunoconjugates and chemotherapeutic agents

ABSTRACT

The present invention is based on the discovery that the administration of at least one immunoconjugate and at least one chemotherapeutic agent provides an unexpectedly superior treatment for cancer. The present invention is directed to compositions comprising at least one immunoconjugate and at least one chemotherapeutic agent and to methods of treating cancer using at least one immunoconjugate and at least one chemotherapeutic agent. The present invention also provides methods of modulating the growth of selected cell populations, such as cancer cells, by administering a therapeutically effective amount of at least one chemotherapeutic agent and at least one immunoconjugate.

RELATED APPLICATIONS

Notice: More than one reissue application has been filed for the reissueof U.S. Pat. No. 7,303,749, filed Sep. 29, 2000. The present applicationis a continuation reissue application of U.S. patent application Ser.No. 12/631,508, now abandoned.

This application is a reissue continuation of application Ser. No.12/631,508, filed Dec. 4, 2009, abandoned, which is a ReissueApplication of U.S. application Ser. No. 09/671,995, filed Sep. 29, 2000(U.S. Pat. No. 7,303,749, issued Dec. 4, 2007), which claims priority toU.S. Provisional Application No. 60/157,051, filed Oct. 1, 1999. Theentire disclosures of the aforementioned related applications andpatents are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention is based on the discovery that the administrationof at least one immunoconjugate and at least one chemotherapeutic agentprovides an unexpectedly superior treatment for cancer. The presentinvention is directed to compositions comprising at least oneimmunoconjugate and at least one chemotherapeutic agent and to methodsof treating cancer using a therapeutically effective amount of at leastone immunoconjugate and at least one chemotherapeutic agent. The presentinvention is also directed to methods of modulating the growth ofselected cell populations using a therapeutically effective amount of atleast one chemotherapeutic agent and at least one immunoconjugate.

BACKGROUND OF THE INVENTION

Of all lung cancer cases diagnosed in the United States every year,20-25% are small cell lung cancer (SCLC). Current treatments for smallcell lung cancer include surgery, radiation treatment, andchemotherapeutic agents, such as paclitaxel or a combination ofetoposide and cisplatin. Despite these treatment options, there is onlya 1-5% survival rate after 5 years in patients who have clinicallyevident metastatic disease upon diagnosis. Glisson et al, Journal ofClinical Oncology, 17(8):2309-2315 (August 1999).

Pre-clinical studies reveal that small cell lung cancers can also betreated with an immunoconjugate comprising a monoclonal antibody and amaytansinoid. Liu et al, Proceedings of the American Association forCancer Research, 38:29 (abstract 190) (1997). In this study, themaytansinoid was DM1, and the monoclonal antibody was humanized N901.Humanized monoclonal antibody N901 targets CD56, which is expressed onsubstantially all small cell lung cancers.

There is a need in the art for new and more effective methods fortreating cancer. The present invention is directed to these, as well asother, important ends.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that the use of at leastone chemotherapeutic agent and at least one immunoconjugate producesunexpectedly superior results in the treatment of cancer.

The present invention describes methods of treating cancer in a patientin need thereof by administering to the patient a therapeuticallyeffective amount of at least one chemotherapeutic agent and at least oneimmunoconjugate. The chemotherapeutic agent can be any known in the artincluding, for example, taxane compounds, compounds that act via taxanemechanisms, platinum compounds, epipodophyllotoxin compounds,camptothecin compounds, or any combination thereof. The immunoconjugatecan comprise a cell binding agent and at least one therapeutic agent forkilling selected cell populations. The cell binding agent is preferablya monoclonal antibody or a fragment thereof, and the therapeutic agentfor killing selected cell populations is preferably an anti-mitoticagent, such as a maytansinoid, a Vinca alkaloid, a dolastatin, or acryptophycin. In particularly preferred embodiments, the immunoconjugatecomprises the maytansinoid DM1 and humanized N901 monoclonal antibody.The chemotherapeutic agent and immunoconjugate can be administeredseparately or as components of the same composition.

The present invention also describes methods of modulating the growth ofselected cell populations, such as cancer cells, by administering atherapeutically effective amount of at least one chemotherapeutic agentand at least one immunoconjugate. The chemotherapeutic agent can be anyknown in the art including, for example, taxane compounds, compoundsthat act via taxane mechanisms, platinum compounds, epipodophyllotoxincompounds, camptothecin compounds, or any combination thereof. Theimmunoconjugate can comprise a cell binding agent and at least onetherapeutic agent for killing selected cell populations. The cellbinding agent is preferably a monoclonal antibody or a fragment thereof,and the therapeutic agent for killing selected cell populations ispreferably an anti-mitotic agent, such as a maytansinoid, a Vincaalkaloid, a dolastatin, or a cryptophycin. In particularly preferredembodiments, the immunoconjugate comprises the maytansinoid DM1 andhumanized N901 monoclonal antibody. The chemotherapeutic agent andimmunoconjugate can be administered separately or as components of thesame composition.

The present invention also describes compositions comprising at leastone chemotherapeutic agent and at least one immunoconjugate. Thechemotherapeutic agent can be any known in the art including, forexample, taxane compounds, compounds that act via taxane mechanisms,platinum compounds, epipodophyllotoxin compounds, camptothecincompounds, or any combination thereof. The immunoconjugate can comprisea cell binding agent and at least one therapeutic agent for killingselected cell populations. The cell binding agent is preferably amonoclonal antibody or a fragment thereof, and the therapeutic agent forkilling selected cell populations is preferably an anti-mitotic agent,such as a maytansinoid, a Vinca alkaloid, a dolastatin, or acryptophycin. In particularly preferred embodiments, the immunoconjugatecomprises the maytansinoid DM1 and humanized N901 monoclonal antibody.The composition can comprise a pharmaceutically acceptable carrier,excipient or diluent.

These and other aspects of the present invention are described in detailherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows maytansine (1a) and maytansinol (1b).

FIG. 2 shows the synthesis of disulfide-containing derivatives ofN-methyl-L-alanine.

FIG. 3 shows the synthesis of disulfide- and thiol-containingmaytansinoids which can be linked to cell binding agents via a disulfideor any other sulfur-containing link such as thioether or thioesterlinks. The synthesis starts with the intermediates of FIG. 2.

FIG. 4a shows the synthesis of disulfide- and thiol-containingderivatives of N-methyl-L-cysteine.

FIG. 4b shows the synthesis of disulfide- and thiol-containingmaytansinoids from the intermediates of FIG. 4a that can be conjugatedto cell binding agents via a disulfide or any other sulfur-containinglink such as thioether or thioester links.

FIG. 5 is a graph comparing the anti-tumor activity of (i) a control,(ii) huN901-DM1, (iii) paclitaxel, and (iv) the combination ofhuN901-DM1 and paclitaxel, against small cell lung cancer xenografts inSCID mice.

FIG. 6 is a graph comparing the anti-tumor activity of (i) a control,(ii) huN901-DM1, (iii) the combination of cisplatin and etoposide, and(iv) the combination of huN901-DM1, cisplatin and etoposide, againstsmall cell lung cancer xenografts in SCID mice.

FIG. 7 is a graph comparing the anti-tumor activity of (i) a control,(ii) huN901-DM1, (iii) docetaxel, and (iv) the combination of huN901-DM1and docetaxel, against small cell lung cancer xenografts in SCID mice.

FIG. 8 is a graph comparing the anti-tumor activity of (i) a control,(ii) huN901-DM1, (iii) topotecan, and (iv) the combination of huN901-DM1and topotecan, against small cell lung cancer xenografts in SCID mice.

FIG. 9 is a graph comparing the anti-tumor activity of (i) a control,(ii) huC242-DM1, (iii) paclitaxel, and (iv) the combination ofhuC242-DM1 and paclitaxel, against human lung adenocarcinoma xenograftsin SCID mice.

FIG. 10 is a graph comparing the anti-tumor activity of (i) a control,(ii) huC242-DM1, (iii) CPT-11 (also called irinotecan), and (iv) thecombination of huC242-DM1 and CPT-11, against human colon cancerxenografts in SCID mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the unexpected discovery that theadministration of at least one chemotherapeutic agent and at least oneimmunoconjugate produces superior results in the treatment of cancer.Appropriate chemotherapeutic agents and immunoconjugates are describedherein.

The immunoconjugates of the present invention comprise at least onetherapeutic agent for killing selected cell populations linked to a cellbinding agent. The therapeutic agent for killing selected cellpopulations is preferably an anti-mitotic agent. Anti-mitotic agents,which are known in the art, kill cells by inhibiting tubulinpolymerization and, therefore, microtubule formation. Any anti-mitoticagent known in the art can be used in the present invention, including,for example, maytansinoids, Vinca alkaloids, dolastatins, cryptophycins,and/or any other agent that kills cells by inhibiting tubulinpolymerization. Preferably, the anti-mitotic agent is a maytansinoid.

Maytansinoids that can be used in the present invention, to produce themodified maytansinoid capable of being linked to a cell binding agent,are well known in the art and can be isolated from natural sourcesaccording to known methods or prepared synthetically according to knownmethods. Preferred maytansinoids are those described, for example, inU.S. Pat. No. 5,208,020, the disclosure of which is incorporated byreference herein in its entirety.

Suitable maytansinoids include maytansinol and maytansinol analogues.Examples of suitable maytansinol analogues include those having amodified aromatic ring and those having modifications at otherpositions. Specific examples of suitable analogues of maytansinol havinga modified aromatic ring include: C-19-dechloro (U.S. Pat. No.4,256,746) (prepared by LAH reduction of ansamitocin P-2); C-20-hydroxy(or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos. 4,361,650 and4,307,016) (prepared by demethylation using Streptomyces or Actinomycesor dechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (—OCOR),+/−dechloro (U.S. Pat. No. 4,294,757) (prepared by acylation using acylchlorides).

Specific examples of suitable analogues of maytansinol havingmodifications of other positions include: C-9-SH (U.S. Pat. No.4,424,219) (prepared by the reaction of maytansinol with H₂S or P₂S₅);C-14-alkoxymethyl(demethoxy/CH₂OR) (U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia); C-15-hydroxy/acyloxy (U.S. Pat. No.4,364,866) (prepared by the conversion of maytansinol by Streptomyces);C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudlflora); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and4,322,348) (prepared by the demethylation of maytansinol byStreptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by thetitanium trichloride/LAH reduction of maytansinol).

In order to link the maytansinoid to the cell binding agent, themaytansinoid must be modified, and a linking group can be used. Suitablelinking groups are known in the art and include, for example, disulfidegroups, thioether groups, acid labile groups, photolabile groups,peptidase labile groups and esterase labile groups. Preferred aredisulfide groups and thioether groups.

The linking group is part of a chemical moiety that is covalently boundto the maytansinoid through conventional methods. In a preferredembodiment, the chemical moiety can be covalently bound to themaytansinoid via an ester linkage.

Many positions on maytansinoids are useful as the linkage position,depending upon the type of link. For example, for forming an esterlinkage, the C-3 position having a hydroxyl group, the C-14 positionmodified with hydroxymethyl, the C-15 position modified with hydroxy,and the C-20 position having a hydroxy group are all expected to beuseful. The C-3 position is preferred and the C-3 position ofmaytansinol is especially preferred. Also preferred is anN-methyl-alanine-containing C-3 ester and anN-methyl-cysteine-containing C-3 ester of maytansinol or its analogues.

The synthesis of esters of maytansinol having a linking group isdescribed in U.S. Pat. No. 5,208,020. While the synthesis of esters ofmaytansinol having a linking group is described herein in terms of thioland disulfide linking groups, one of skill in the art will understandthat other linking groups can also be used with the invention, as canother maytansinoids.

The synthesis of maytansinoid derivatives can be described by referenceto FIGS. 1, 2, 3, 4a and 4b, where disulfide-containing maytansinoidesters are prepared by condensing maytansinol 1b with freshly preparedN-methyl-L-alanine or N-methyl-L-cysteine derivatives containing adisulfide group.

ω-Mercapto-carboxylic acids of varying chain lengths are converted intotheir respective methyl-dithio, e.g., 3a-3d (where n=1-10, includingbranched and cyclic aliphatics), or aryl-dithio, e.g., 4a-4b,derivatives by reacting them with methyl methanethiolsulfonate oraryldisulfides, such as diphenyldisulfide and ring substituteddiphenyldisulfides and heterocyclic disulfides such as2,2-dithiopyridine. The carboxylic acids are activated and then reactedwith N-methyl-L-alanine to form the desired carboxylic acid compounds,e.g., 5a-5f, for condensation with maytansinol 1b.

Esterification of maytansinol 1b or an analogue with the carboxylicacids 5a-5f gives the disulfide-containing maytansinoids 6a-6f. Cleavageof the disulfide group in 6a-6f with dithiothreitol gives thethiol-containing maytansinoids 7a-7c, which are readily linked viadisulfide or thioether links to cell binding agents. N-methyl-L-alaninecan be prepared as described in the literature (Fu et al, J. Amer. Chem.Soc., 75:1953); or is obtainable commercially (Sigma Chemical Company).

In another embodiment, N-methyl-cysteine or N-methylhomocysteine can beconverted to the respective disulfide derivatives 8 (n=1 and 2,respectively) which are then acylated to yield the desired carboxylicacids 9 (n=1 and 2, respectively). Maytansinol is then esterified with 9(n=1) to give disulfide-containing ester 10. Reduction of 10a withdithiothreitol as described for 7b produces the thiol-containingmaytansinoid 11 which can be conjugated to cell binding agents.N-methyl-cysteine can be prepared as described in Undheim et al, ActaChem. Scand., 23:3129-3133 (1970).

More specifically, maytansinol 1b is derived from maytansine 1a or otheresters of maytansinol by reduction such as with lithium aluminumhydride. (Kupchan et al, J. Med. Chem., 21:31-37 (1978); U.S. Pat. No.4,360,462). It is also possible to isolate maytansinol from themicroorganism Nocardia (U.S. Pat. No. 4,151,042). Maytansinol is thenconverted to the different ester derivatives, 6a to 6f and 10, using asuitable agent such as dicyclohexylcarbodiimide (DCC) and catalyticamounts of zinc chloride (U.S. Pat. Nos. 4,137,230 and 4,260,609; Kawaiet al, Chem. Pharm. Bull., 32:3441-3951 (1984)). The two diastereomericproducts containing the D and L-aminoacyl side chains result. Thediastereomeric maytansinoid esters are readily separated by preparativeTLC on silica gel. For example, using Analtech GF plates (1000 microns)and developing with 6% methanol in chloroform yields distinct banding:the desired bands are scraped off the plate and the products extractedwith ethyl acetate (Kupchan, J. Med. Chem., 21:31-37 (1978) and U.S.Pat. No. 4,360,462).

Reduction of the disulfide-containing maytansinoids to the correspondingmercapto-maytansinoids 7a, 7b, 7c and 11, is achieved by treatment withdithiothreitol (DTT) and purification by HPLC using a Waters radialpakC-18 column and eluting with a linear gradient of 55% to 80%acetonitrile in H₂O over 10 minutes at a flow rate of 1.5 ml/min.

When analogues of maytansinol are used as the starting material to giveanalogous disulfide-containing maytansinoid esters, the analogues areprepared before reacting them with the N-methyl-L-alanine orN-methyl-L-cysteine derivatives.

One example of N-methyl-alanine-containing maytansinoid derivativesuseful in the present invention is represented by formula (I):

wherein

Z₀ represents H or SR, wherein R represents methyl, linear alkyl,branched alkyl, cyclic alkyl, simple or substituted aryl orheterocyclic;

p represents an integer of 1 to 10; and

“may” represents a maytansinoid.

In a preferred embodiment of the compound of formula (I), Z₀ representsSR, R represents methyl, and p represents an integer of 2.

Another example of N-methyl-alanine-containing maytansinoid derivativesuseful in the present invention is represented by formula (II):

wherein

R₁ and R₂, which may be the same or different, represents H, CH₃ orCH₂CH₃;

Z₁ represents H or SR³, wherein R³ represents methyl, linear alkyl,branched alkyl, cyclic alkyl, simple or substituted aryl, orheterocyclic:

m represents 0, 1, 2 or 3; and

“may” represents a maytansinoid.

Another example of N-methyl-alanine-containing maytansinoid derivativesuseful in the present invention is represented by formula (III):

wherein:

Z₂ represents H or SR₄, wherein R₄ represents methyl, linear alkyl,branched alkyl cyclic alkyl, simple or substituted aryl, orheterocyclic;

n represents an integer of 3 to 8; and

“may” represents a maytansinoid.

Yet another example of N-methyl-alanine-containing maytansinoidderivatives useful in the present invention is represented by formula(IV):

wherein:

Z₀ represents H or SR, wherein R represents methyl, linear alkyl,branched alkyl, cyclic alkyl, simple or substituted aryl orheterocyclic:

t represents 1, 2 or 3;

Y₀ represents Cl or H; and

X₃ represents H or CH₃.

A specific example of N-methyl-cysteine-containing maytansinoidderivatives useful in the present invention is represented by formula(V):

wherein:

Z₃ represents H or SR₅, wherein R₅ represents methyl, linear alkyl,branched alkyl, cyclic alkyl, simple or substituted aryl, orheterocyclic;

o represents 1, 2 or 3;

p represents 0 or an integer of 1 to 10; and

“may” represents a maytansinoid.

Another specific example of N-methyl-cysteine-containing maytansinoidderivatives useful in the present invention is represented by formula(VI):

wherein:

Z₃ represents H or SR₅, wherein R₅ represents methyl, linear alkyl,branched alkyl, cyclic alkyl, simple or substituted aryl orheterocyclic;

o represents 1, 2, or 3;

q represents 0 or an integer of 1 to 10;

Y₀ represents Cl or H; and

X₃ represents H or CH₃.

Examples of linear alkyls include methyl, ethyl, propyl, butyl, pentyl,and hexyl. Examples of branched alkyls include isopropyl, isobutyl,sec-butyl, tert-butyl, isopentyl, and 1-ethyl-propyl. Examples of cyclicalkyls include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.Examples of simple aryls include phenyl, and naphthyl. Examples ofsubstituted aryls include aryls such as those described abovesubstituted with alkyl groups, with halogens, such as Cl, Br, F, nitrogroups, amino groups, sulfonic acid groups, carboxylic acid groups,hydroxy groups, and alkoxy groups. Examples of heterocyclics arecompounds wherein the heteroatoms are selected from O, N and S, andinclude pyrrollyl, pyridyl, furyl, and thiophene.

Vinca alkaloids that can be used in the present invention, to producethe modified Vinca alkaloids capable of being linked to a cell bindingagent, are well known in the art. Such Vinca alkaloids include, forexample, those described in Cancer Principles and Practice in Oncology,4th Ed., DeVita et al, eds., J. B. Lippincott Company, Philadelphia Pa.(1993) and by Morris et al, J. Clin. Oncol., 16:1094-1098 (1998), thedisclosures of which are incorporated herein by reference in theirentirety. Exemplary Vinca alkaloids include vincristine, vinblastine,vindesine, navelbine (vinorelbine), and the like. Other Vinca alkaloidsthat can be used in the present invention include those described, forexample, in U.S. Pat. Nos. 5,369,111, 4,952,408, 5,395,610, 4,522,750,5,888,537, 5,891,724, 5,795,589, 4,172,077, 5,714,163, 5,436,243,3,932,417, 5,869,620, 5,795,575, 5,780,446, 5,676,978, 5,604,237,5,171,217, 4,831,038, 4,828,831, 4,765,972, 4,375,432, 4,309,415,5,939,455, 5,874,402, 5,767,260, 5,763,733, 5,728,687, 5,716,928,5,660,827, 5,541,232, 5,346,897, 5,220,016, 5,208,238, 5,190,949,4,479,957, 4,160,767, 4,159,269, 4,096,148, RE 30,561, RE 30,560, U.S.Pat. Nos. 5,935,955, 5,922,340, 5,886,025, 5,866,679, 5,863,538,5,855,866, 5,817,321, 5,783,178, 5,776,427, 5,767,110, 5,753,507,5,723,625, 5,698,178, 5,686,578, 5,667,764, 5,654,287, 5,646,124,5,635,515, 5,635,218, 5,606,017, 5,597,830, 5,595,756, 5,583,052,5,561,136, 5,547,667, 5,543,152, 5,529,076, 5,491,285, 5,482,858,5,455,161, 5,430,026, 5,403,574, 5,399,363, 5,397,784, 5,387,578,5,364,843, 5,300,282, 5,182,368, 5,162,115, 5,147,294, 5,108,987,5,100,881, 5,047,528, 5,030,620, 5,004,593, 4,946,833, 4,931,468,4,923,876, 4,801,688, 4,737,586, 4,667,030, 4,617,305, 4,578,351,4,476,026, 4,399,069, 4,279,817, 4,208,414, 4,199,504, 4,070,358,4,029,663, 3,965,254, 3,954,773, 3,944,554, 3,887,565, 6,120,800,6,071,947, 6,071,930, 6,069,146, 6,063,911, 5,994,367, 5,962,216, and5,945,315, the disclosures of which are incorporated by reference hereinin their entirety.

The Vinca alkaloids can be linked to cell binding agents, such asantibodies, via acid-labile hydrazide links by methods described by, forexample, Laguzza et al, J. Med. Chem., 32:548-555 (1989), Schrappe etal, Cancer Res., 52:3838-3844 (1992), and Apelgren et al, Cancer Res.,50:3540-3544 (1990), the disclosures of which are incorporated byreference herein in their entirety. A preferable method is to link theVinca alkaloids to a cell binding agent via disulfide bonds. The carboxyester at the C-3 position of vinblastine, vincristine and navelbine canbe hydrolzyed to the corresponding carboxylic acid using standardchemical methods. In vindesine, the carboxamide group at C-3 can behydrolyzed to the free carboxy group. The free carboxy group in each ofthe Vinca alkaloids can be converted to an amide compound containing aterminal disulfide group by reaction with a protected cysteamine (e.g.,methydithiocysteamine) in the presence of a coupling agent such asdicyclohexyl-carbodidimide (DCC) or ethyldimethylamin-propylcarbodiimide (EDC). The resulting disulfidecontaining Vinca alkaloid is reduced with a reducing agent, such asdithiothreitol, to provide a thiol-containing compound. Thethiol-containing Vinca alkaloid can be coupled to a cell-binding agentvia disulfide exchange as described herein for the preparation ofantibody-maytansinoid conjugates.

Dolastatins that can be used in the present invention, to produce themodified dolastatins capable of being linked to a cell binding agent,are well known in the art. Such dolastatins include, for example, thosedescribed by Pitot et al, Clin. Cancer Res., 5:525-531 (1999) andVillalona-Calero et al, J. Clin. Oncol., 16:2770-2779 (1998), thedisclosures of which are incorporated by reference herein in theirentirety. Exemplary dolastatins include dolastatin 10, dolastatin 15,and the like. Other dolastatins that can be used in the presentinvention include those described, for example, in U.S. Pat. Nos.5,945,543, 5,939,527, 5,886,147, 5,886,025, 5,883,120, 5,856,324,5,840,699, 5,831,002, 5,821,222, 5,807,984, 5,780,588, 5,767,237,5,750,713, 5,741,892, 5,665,860, 5,663,149, 5,654,399, 5,635,483,5,626,864, 5,599,902, 5,554,725, 5,530,097, 5,521,284, 5,504,191,5,502,032, 5,410,024, 5,410,024, 5,378,803, 5,352,804, 5,138,036,5,091,368, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444,4,486,414, 4,414,205, 6,103,913, 6,103,698, 6,096,757, 6,034,065,6,020,495, 6,017,890, 6,004,934, 5,985,837, 5,965,700, and 5,965,537,the disclosures of which are incorporated by reference herein in theirentirety.

The synthetic scheme described for dolastatin 10 by Pettit et al, J. Am.Chem. Soc., 111:5463-5465 (1989), the disclosure of which isincorporated by reference herein in its entirety, can be followed, withminor modification, to provide a thiol-containing dolastatin that can belinked via disulfide bonds to a cell binding agent, such as an antibody.The phenylalanine moiety in the dolphenine residue in the C-terminal ofdolastatin 10 is replaced by a methyldithio-substituent containing aminoacid. Thus, tyrosine can be converted into an ether by reaction with acommercially available dibromoalkane, such as 1,3-dibromobutane, usingstandard chemical methods. The resulting bromo compound is reacted withpotassium thioacetate, followed by hydrolysis, to give athiol-containing tyrosine. Conversion is achieved as described byPettit, supra. The thiol-containing dolastatin can be coupled to a cellbinding agent via disulfide exchange as described herein for thepreparation of an antibody-maytansinoid conjugate.

Cryptophycins that can be used in the present invention, to produce themodified cryptophycins capable of being linked to a cell binding agent,are well known in the art. Such cryptophycins include, for example,those described by Smith et al, Cancer Res., 54:3779-3783 (1994), Pandaet al, Proc. Natl. Acad. Sci., 95:9313-9318 (1998), and Bai et al,Cancer Res., 56:4398-4406 (1996), the disclosures of which areincorporated by reference herein in their entirety. Exemplarycryptophycins include cryptophycin 52, cryptophycin 1, and the like.Other cryptophycins that can be used in the present invention includethose described, for example, in Great Britain Patent No. 2220657;European Patent Nos. 870506, 870501, 861838, 861839, 792875 and 870510;U.S. Pat. Nos. 6,103,913, 6,046,177, 6,020,512, 6,013,626, 5,977,387,5,955,423, 5,952,298, 5,945,315, 5,886,025, and 5,833,994; and WIPOPublication Nos. 98/38178, 98/38164, 98/08829, 98/08506, 98/08505,97/31632, 97/08334, 97/07798, 98/09601, 97/23211, 98/46581, 98/38158,98/09988, 98/09974, 98/08812, and 98/09955, the disclosures of which areincorporated herein by reference in their entirety.

The aromatic methoxy group in the cryptophycins can be hydrolyzed bystandard chemical or enzymatic methods to give the phenolic derivative.The phenol group can be converted into an ether by reaction with acommercially available dibromoalkane, such as 1,3-dibromobutane, usingstandard chemical methods. The resulting bromo compound is reacted withpotassium thioacetate, followed by hydrolysis, to give athiol-containing cryptophycin. The thiol-containing cryptophycin can becoupled to a cell binding agent via disulfide exchange as describedherein for the preparation of antibody-maytansinoid conjugates.

Disulfide-containing and mercapto-containing maytansinoid (or Vincaalkaloid or dolastatin or cryptophycin) drugs of the invention can beevaluated for their ability to suppress proliferation of variousunwanted cell lines using in vitro methods generally accepted in the artas being predictive of in vivo activity. For example, cell lines such asthe human epidermoid carcinoma line KB, the human breast tumor lineSKBR3 and the Burkitt's lymphoma line Namalwa can easily be used for theassessment of cytotoxicity of these compounds. Cells to be evaluated canbe exposed to the compounds for 24 hours and the surviving fractions ofcells measured in direct assays by known methods. IC₅₀ values can thenbe calculated from the results of the assays.

The effectiveness of the immunoconjugates as therapeutic agents dependson the careful selection of an appropriate cell binding agent. Cellbinding agents may be of any kind presently known, or that become known,and include peptides and non-peptides. Generally, these can beantibodies (especially monoclonal antibodies), lymphokines, hormones,growth factors, nutrient-transport molecules (such as transferrin), orany other cell binding molecule or substance.

More specific examples of cell binding agents that can be used include:monoclonal antibodies; fragments of antibodies such as Fv, Fab, Fab′,and F(ab′)₂ (Parham, J. Immunol., 131:2895-2902 (1983); Spring et al, J.Immunol., 113:470-478 (1974); Nisonoff et al, Arch. Biochem. Biophys.,89:230-244 (1960)); interferons (e.g., α, β, γ); lymphokines such asIL2, IL3, IL-4, IL-6; hormones such as insulin, TRH (thyrotropinreleasing hormone), MSH (melanocyte-stimulating hormone), steroidhormones such as androgens and estrogens; growth factors andcolony-stimulating factors such as EGF, TGF-α, G-CSF, M-CSF and GM-CSF(Burgess, Immunology Today, 5:155-158 (1984)); and transferrin (O'Keefeet al, J. Biol. Chem., 260:932-937 (1985)).

Monoclonal antibody techniques allow for the production of extremelyspecific cell binding agents in the form of specific monoclonalantibodies. Particularly well known in the art are techniques forcreating monoclonal antibodies produced by immunizing mice, rats,hamsters or any other mammal with the antigen of interest such as theintact target cell, antigens isolated from the target cell, whole virus,attenuated whole virus, and viral proteins such as viral coat proteins.Sensitized human cells can also be used.

Selection of the appropriate cell binding agent is a matter of choicethat depends upon the particular cell population that is to be targeted,but in general monoclonal antibodies are preferred if an appropriate oneis available.

For example, the monoclonal antibody J5 is a murine IgG_(2a) antibodythat is specific for the Common Acute Lymphoblastic Leukemia Antigen(CALLA) (Ritz et al, Nature, 283:583-585 (1980)) and can be used if thetarget cells express CALLA such as in the disease of acute lymphoblasticleukemia. Similarly, the monoclonal antibody anti-B4 is a murine IgG₁,that binds to the CD19 antigen on B cells (Nadler et al, J. Immunol.,131:244-250 (1983)) and can be used if the target cells are B cells ordiseased cells that express this antigen such as in non-Hodgkin'slymphoma or chronic lymphoblastic leukemia.

Additionally, GM-CSF which binds to myeloid cells can be used as a cellbinding agent to diseased cells from acute myelogenous leukemia. IL-2which binds to activated T-cells can be used for prevention oftransplant graft rejection, for therapy and prevention ofgraft-versus-host disease, and for treatment of acute T-cell leukemia.MSH which binds to melanocytes can be used for the treatment ofmelanoma.

Cancers of the breast and testes can be successfully targeted withestrogen (or estrogen analogues) or androgen (or androgen analogues),respectively, as cell binding agents.

In a preferred embodiment, the antibody or fragment thereof is one thatis specific for lung cancer, preferably small cell lung cancer. Anantibody or fragment thereof that is specific for small cell lung cancercan be determined by methods described in the art, such as by Doria etal, Cancer 62:1939-1945 (1988). Preferably, the antibody or fragmentthereof binds to an epitope on the CD56 antigen, which is expressed onsubstantially all small cell lung cancers. For example, N901 is an IgG1murine monoclonal antibody (also called anti-N901) that is reactive withCD56, which is expressed on tumors of neuroendocrine origin, such assmall cell lung cancer. See Griffin et al, J. Immunol. 130:2947-2951(1983), and Roguska et al, Proc. Natl. Acad. Sci. USA, 91:969-973(1994), the disclosure of which are incorporated by reference herein intheir entirety.

Preferred antibodies or fragments thereof that are specific for smallcell lung cancers include, but are not limited to, N901, NKH-1, Leu-7,anti-Leu-7, and the like (Doria et al, Cancer 62:1939-1945 (1988);Kibbelaar et al, Journal of Pathology, 159:23-28 (1989)). Other suitableantibodies or fragments thereof for use in the present inventioninclude, for example, S-L 3-5, S-L 4-20, S-L 7-3, S-L11-14, TFS-4,MOC-1, MOC-21, MOC-31, MOC-32, MOC-52, 123A8, 123C3, UJ13A, B10/B12,SWA4, SWA20, SWA21, SWA22, SWA23, LAM-8, 534F8, 703D4704A1 and SM1,which are further described in Table I of Chapter 3 of the Proceedingsof the First International Workshop on Small Cell Lung Cancer Antigens(London 1987), published in Lung Cancer, 4:15-36 (1988), the disclosuresof which are incorporated by reference herein in their entirety. In amost preferred embodiment, the antibody is N901, or a fragment thereof,that binds to an epitope on the CD56 antigen, such as Fv, Fab, Fab′ andF(ab′)₂. The monoclonal antibody or fragment thereof can be any otherantibody that binds to the CD56 antigen with the same specificity asN901. “Same specificity” means that the antibody or fragment thereof canbind to the same antigen as demonstrated by a competitive binding assaywith N901.

Another preferred antibody or fragment thereof that is useful in thepresent invention is C242 (commercially available from CanAg DiagnosticsAB, Sweden). C242 is also described in U.S. Pat. No. 5,552,293, thedisclosure of which is incorporated by reference herein in its entirety.

In other preferred embodiments, the antibodies described herein arehumanized antibodies or fragments thereof because humanized antibodiesor fragments thereof are not expected to elicit an immune response inhumans. Generally, antibodies can be humanized through the applicationof different humanization technologies described, for example, in U.S.Pat. Nos. 5,225,539, 5,585,089, and 5,639,641, the disclosures of whichare incorporated by reference herein in their entirety. The preparationof different versions of humanized N901, is described, for example, byRoguska et al Proc. Natl. Acad. Sci. USA, 91:969-973 (1994), and Roguskaet al, Protein Eng., 9:895:904 (1996), the disclosures of which areincorporated by reference herein in their entirety. To denote ahumanized antibody, the letters “hu” or “h” appear before the name ofthe antibody. For example, humanized N901 is also referred to as huN901or hN901.

Conjugates of the maytansinoid derivatives of the invention and a cellbinding agent can be formed using any techniques presently known orlater developed. The maytansinoid ester can be modified to yield a freeamino group and then linked to an antibody or other cell binding agentvia an acid-labile linker, or a photolabile linker. The maytansinoidester can be condensed with a peptide and subsequently linked to a cellbinding agent to produce a peptidase-labile linker. The maytansinoidester can be treated to yield a primary hydroxyl group, which can besuccinylated and linked to a cell binding agent to produce a conjugatethat can be cleaved by intracellular esterases to liberate free drug.Most preferably, the maytansinoid esters are treated to create a free orprotected thiol group, and then one or many disulfide orthiol-containing maytansinoid derivatives are covalently linked to thecell binding agent via disulfide bond(s).

Representational conjugates of the invention are antibody/maytansinoidderivatives, antibody fragment/maytansinoid derivatives, epidermalgrowth factor (EGF)/maytansinoid derivatives, melanocyte stimulatinghormone (MSH)/maytansinoid derivatives, thyroid stimulating hormone(TSH)/maytansinoid derivatives, estrogen/maytansinoid derivatives,estrogen analogue/maytansinoid derivatives, androgen/maytansinoidderivatives, androgen analogue/maytansinoid derivatives.

Maytansinoid conjugates of antibodies, antibody fragments, proteinhormones, protein growth factors and other proteins are made in the sameway. For example, peptides and antibodies can be modified withcrosslinking reagents such as N-succinimidyl3-(2-pyridyldithio)propionate, N-succinimidyl4-(2-pyridyldithio)-pentanoate (SPP),4-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT),N-succinimidyl-3-(2-pyridyldithio)-butyrate (SDPB), 2-iminothiolane, oracetylsuccinic anhydride by known methods (U.S. Pat. No. 4,563,304;Carlsson et al, Biochem. J., 173:723-737 (1978); Blättler et al,Biochem., 24:1517-1524 (1985); Lambert et al, Biochem., 22:3913-3920(1983); Klotz et al, Arch. Biochem. Biophys., 96:605 (1962); and Liu etal, Biochem., 18:690 (1979), Blakey and Thorpe, Antibody,Immunoconjugates and Radiopharmaceuticals, 1:1-16 (1988); Worrell et al,Anti-Cancer Drug Design, 1:179-184 (1986), the disclosures of which areincorporated by reference herein in their entirety). The cell bindingagent containing free or protected thiol groups thus-derived is thenreacted with a disulfide- or thiol-containing maytansinoid to produceconjugates. The conjugates can be purified by HPLC or by gel filtration.

Similarly, for example, estrogen and androgen cell binding agents, suchas estradiol and androstenediol, can be esterified at the C-17 hydroxygroup with an appropriate disulfide-containing carboxylic acid, e.g.,dicyclohexylcarbodiimide, as a condensing agent. Examples of suchcarboxylic acids that can be used are 3-(2-pyridyldithio)propanoic acid,3-methyldithiopropanoic acid, 3-phenyldithio-propanoic acid, and4-(2-pyridyldithio)pentanoic acid. Esterification of the C-17 hydroxygroup can also be achieved by reaction with an appropriately protectedthiol group-containing carboxylic acid chloride, such as3S-acetylpropanoyl chloride. Other methods of esterification can also beused as described in the literature (Haslam, Tetrahedron, 36:2400-2433(1980)). The protected or free thiol-containing androgen or estrogen canthen be reacted with a disulfide or thiol-containing maytansinoid toproduce conjugates. The conjugates can be purified by columnchromatography on silica gel or by HPLC.

Preferably monoclonal antibody or cell binding agent/maytansinoidconjugates are those that are joined via a disulfide bond, as discussedabove, that are capable of delivering maytansinoid molecules. Such cellbinding conjugates are prepared by known methods such as modifyingmonoclonal antibodies with succinimidyl pyridyl-dithiopropionate (SPDP)or SPP (Carlsson et al, Biochem. J., 173:723-737 (1978)). The resultingthiopyridyl group is then displaced by treatment with thiol-containingmaytansinoids to produce disulfide linked conjugates. Alternatively, inthe case of the aryldithio-maytansinoids, the formation of the cellbinding conjugate is effected by direct displacement of the aryl-thiolof the maytansinoid by sulfhydryl groups previously introduced intoantibody molecules. Conjugates containing 1 to 10 maytansinoid drugslinked via a disulfide bridge are readily prepared by either method.

More specifically, a solution of the dithiopyridyl modified antibody ata concentration of 2.5 mg/ml in 0.05 M potassium phosphate buffer and0.05 M sodium chloride, at pH 6.5 containing 2 mM EDTA is treated withthe thiol-containing maytansinoid (1.7 molar equivalent/dithiopyridylgroup). The release of pyridine-2-thione from the modified antibody ismonitored spectrophotometrically at 343 nm. The reaction is allowed toproceed up to 18 hours. The antibody-maytansinoid conjugate is purifiedand freed of unreacted drug and other low molecular weight material bygel filtration through a column of Sephacryl S-300. The number ofmaytansinoids bound per antibody molecule can be determined by measuringthe ratio of the absorbance at 252 nm and 280 nm. An average of 1-10maytansinoid molecules/antibody molecule can be linked via disulfidebonds by this method.

Antibody-maytansinoid conjugates with non-cleavable links can also beprepared. The antibody can be modified with crosslinking reagents suchas succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC),sulfo-SMCC, maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBSor succinimidyl-iodoacetate, as described in the literature, tointroduce 1-10 reactive groups (Yoshitake et al, Eur. J. Biochem.,101:395-399 (1979); Hashida et al, J. Applied Biochem., 56-63 (1984);and Liu et al, Biochem., 18:690-697 (1979)). The modified antibody isthen reacted with the thiol-containing maytansinoid derivative toproduce a conjugate. The conjugate can be purified by gel filtrationthrough a Sephacryl S-300 column.

The modified antibodies are treated with the thiol-containingmaytansinoid (1.25 molar equivalent/maleimido group). The mixtures areincubated overnight at about 4° C. The antibody-maytansinoid conjugatesare purified by gel filtration through a Sephadex G-25 column.Typically, an average of 1-10 maytansinoids per antibody are linked.

A preferred method is to modify antibodies with succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) to introducemaleimido groups followed by reaction of the modified antibody with athiol-containing maytansinoid to give a thioether-linked conjugate.Again conjugates with 1 to 10 drug molecules per antibody moleculeresult.

As described herein, the present invention is based on the unexpecteddiscovery that the use of at least one immunoconjugate and at least onechemotherapeutic agent produces superior results in treating cancer. Anychemotherapeutic agent known in the art can be used in combination withthe immunoconjugate of the present invention to achieve the unexpectedlysuperior results described and demonstrated herein. Preferably, thechemotherapeutic agent is a taxane compound, a compound that acts via ataxane mechanism, a platinum compound, an epidophyllotoxin compound, acamptothecin compound, and/or any combination thereof. As is known inthe art, platinum compounds and epidophyllotoxin compounds are generallyused together for treating cancer.

In one embodiment, the present invention provides methods of treatingcancer and/or modulating the growth of selected cell populations (e.g.,cancer cells) by administering at least one immunoconjugate and at leastone taxane compound. In another embodiment, the present inventionprovides methods of treating cancer and/or modulating the growth ofselected cell populations (e.g., cancer cells) by administering at leastone immunoconjugate and at least one compound that acts via a taxanemechanism. In another embodiment, the present invention provides methodsof treating cancer and/or modulating the growth of selected cellpopulations (e.g., cancer cells) by administering at least oneimmunoconjugate, and at least one platinum compound. In anotherembodiment, the present invention provides methods of treating cancerand/or modulating the growth of selected cell populations (e.g., cancercells) by administering at least one immunoconjugate, at least oneplatinum compound, and at least one epidophyllotoxin compound. Inanother embodiment, the present invention provides methods of treatingcancer and/or modulating the growth of selected cell populations (e.g.,cancer cells) by administering at least one immunoconjugate and at leastone camptothecin compound. In another embodiment, the present inventionprovides methods of treating cancer and/or modulating the growth ofselected cell populations (e.g., cancer cells) by administering at leastone immunoconjugate and at least one compound that is capable ofinhibiting DNA topoisomerase I. In yet another embodiment, the presentinvention provides methods of treating cancer and/or modulating thegrowth of selected cell populations (e.g., cancer cells) byadministering at least one immunoconjugate, at least one taxanecompound, and at least one platinum compound. In yet another embodiment,the present invention provides methods of treating cancer and/ormodulating the growth of selected cell populations (e.g., cancer cells)by administering at least one immunoconjugate, at least one taxanecompound, at least one platinum compound, and, optionally, at least oneepidophyllotoxin compound. In yet another embodiment, the presentinvention provides methods of treating cancer and/or modulating thegrowth of selected cell populations (e.g., cancer cells) byadministering at least one immunoconjugate, at least one camptothecincompound, at least one platinum compound and, optionally, at least oneepidophyllotoxin compound. In yet another embodiment, the presentinvention provides methods of treating cancer and/or modulating thegrowth of selected cell populations (e.g., cancer cells) byadministering at least one immunoconjugate, at least one compound thatacts via a taxane mechanism and at least one camptothecin compound. Oneskilled in the art will appreciate that the methods described in thepresent invention encompass administering at least one immunoconjugatewith one or more chemotherapeutic agents selected from the groupconsisting of taxane compounds, compounds that act through a taxanemechanism, platinum compounds, epidophyllotoxin compounds, camptothecincompounds and compounds that can inhibit DNA topoisomerase I. In themethods of the present invention, the immunoconjugate andchemotherapeutic agent can be administered simultaneously, about thesame time, or at different times, or can be components of a singlecomposition.

Taxane compounds prevent the growth of cancer cells by affecting cellstructures called microtubules, which play an important role in cellfunctions. In normal cell growth, microtubules are formed when a cellstarts dividing. Once the cell stops dividing, the microtubules arebroken down or destroyed. Taxane compounds stop the microtubules frombreaking down, such that the cancer cells become clogged withmicrotubules so that they cannot grow and divide.

Taxane compounds are known in the art and include, for example,paclitaxel (available as TAXOL® from Bristol-Myers Squibb, Princeton,N.J.), docetaxel (available as TAXOTERE® from Aventis), and the like.Other taxane compounds that become approved by the U.S. Food and DrugAdministration (FDA) or foreign counterparts thereof are also preferredfor use in the methods and compositions of the present invention. Othertaxane compounds that can be used in the present invention include thosedescribed, for example, in 10th NCI-EORTC Symposium on New Drugs inCancer Therapy, Amsterdam, page 100, Nos. 382 and 383 (Jun. 16-19,1998); and U.S. Pat. Nos. 4,814,470, 5,721,268, 5,714,513, 5,739,362,5,728,850, 5,728,725, 5,710,287, 5,637,484, 5,629,433, 5,580,899,5,549,830, 5,523,219, 5,281,727, 5,939,567, 5,703,117, 5,480,639,5,250,683, 5,700,669, 5,665,576, 5,618,538, 5,279,953, 5,243,045,5,654,447, 5,527,702, 5,415,869, 5,279,949, 5,739,016, 5,698,582,5,478,736, 5,227,400, 5,516,676, 5,489,601, 5,908,759, 5,760,251,5,578,739, 5,547,981, 5,547,866, 5,344,775, 5,338,872, 5,717,115,5,620,875, 5,284,865, 5,284,864, 5,254,703, 5,202,448, 5,723,634,5,654,448, 5,466,834, 5,430,160, 5,407,816, 5,283,253, 5,719,177,5,670,663, 5,616,330, 5,561,055, 5,449,790, 5,405,972, 5,380,916,5,912,263, 8,808,113, 5,703,247, 5,618,952, 5,367,086, 5,200,534,5,763,628, 5,705,508, 5,622,986, 5,476,954, 5,475,120, 5,412,116,5,916,783, 5,879,929, 5,861,515, 5,795,909, 5,760,252, 5,637,732,5,614,645, 5,599,820, 5,310,672, RE 34,277, U.S. Pat. Nos. 5,877,205,5,808,102, 5,766,635, 5,760,219, 5,750,561, 5,637,723, 5,475,011,5,256,801, 5,900,367, 5,869,680, 5,728,687, 5,565,478, 5,411,984,5,334,732, 5,919,815, 5,912,264, 5,773,464, 5,670,673, 5,635,531,5,508,447, 5,919,816, 5,908,835, 5,902,822, 5,880,131, 5,861,302,5,850,032, 5,824,701, 5,817,867, 5,811,292, 5,763,477, 5,756,776,5,686,623, 5,646,176, 5,621,121, 5,616,739, 5,602,272, 5,587,489,5,567,614, 5,498,738, 5,438,072, 5,403,858, 5,356,928, 5,274,137,5,019,504, 5,917,062, 5,892,063, 5,840,930, 5,840,900, 5,821,263,5,756,301, 5,750,738, 5,750,562, 5,726,318, 5,714,512, 5,686,298,5,684,168, 5,681,970, 5,679,807, 5,648,505, 5,641,803, 5,606,083,5,599,942, 5,420,337, 5,407,674, 5,399,726, 5,322,779, 4,924,011,5,939,566, 5,939,561, 5,935,955, 5,919,455, 5,854,278, 5,854,178,5,840,929, 5,840,748, 5,821,363, 5,817,321, 5,814,658, 5,807,888,5,792,877, 5,780,653, 5,770,745, 5,767,282, 5,739,359, 5,726,346,5,717,103, 5,710,099, 5,698,712, 5,683,715, 5,677,462, 5,670,653,5,665,761, 5,654,328, 5,643,575, 5,621,001, 5,608,102, 5,606,068,5,587,493, 5,580,998, 5,580,997, 5,576,450, 5,574,156, 5,571,917,5,556,878, 5,550,261, 5,539,103, 5,532,388, 5,470,866, 5,453,520,5,384,399, 5,364,947, 5,350,866, 5,336,684, 5,296,506, 5,290,957,5,274,124, 5,264,591, 5,250,722, 5,229,526, 5,175,315, 5,136,060,5,015,744, 4,924,012, 6,118,011, 6,114,365, 6,107,332, 6,072,060,6,066,749, 6,066,747, 6,051,724, 6,051,600, 6,048,990, 6,040,330,6,030,818, 6,028,205, 6,025,516, 6,025,385, 6,018,073, 6,017,935,6,011,056, 6,005,138, 6,005,138, 6,005,120, 6,002,023, 5,998,656,5,994,576, 5,981,564, 5,977,386, 5,977,163, 5,965,739, 5,955,489,5,939,567, 5,939,566, 5,919,815, 5,912,264, 5,912,263, 5,908,835, and5,902,822, the disclosures of which are incorporated by reference hereinin their entirety.

Other compounds that can be used in the invention are those that actthrough a taxane mechanism. Compounds that act through a taxanemechanism include compounds that have the ability to exertmicrotubule-stabilizing effects and cytotoxic activity against rapidlyproliferating cells, such as tumor cells or other hyperproliferativecellular diseases. Such compounds include, for example, epothilonecompounds, such as, for example, epothilone A, B, C, D, E and F, andderivatives thereof. Other compounds that act through a taxane mechanism(e.g., epothilone compounds) that become approved by the FDA or foreigncounterparts thereof are also preferred for use in the methods andcompositions of the present invention. Epothilone compounds andderivatives thereof are known in the art and are described, for example,in U.S. Pat. Nos. 6,121,029, 6,117,659, 6,096,757, 6,043,372, 5,969,145,and 5,886,026; and WO 97/19086, WO 98/08849, WO 98/22461, WO 98/25929,WO 98/38192, WO 99/01124, WO 99/02514, WO 99/03848, WO 99/07692, WO99/27890, and WO 99/28324, the disclosures of which are incorporatedherein by reference in their entirety.

Other compounds that can be used in the invention include platinumcompounds such as, for example, cisplatin (available as PLATINOL® fromBristol-Myers Squibb, Princeton, N.J.), carboplatin (available asPARAPLATIN® from Bristol-Myers Squibb, Princeton, N.J.), oxaliplatin(available as ELOXATINE® from Sanofi, France), iproplatin, ormaplatin,tetraplatin, and the like. Other platinum compounds that become approvedby the FDA or foreign counterparts thereof are also preferred for use inthe methods and compositions of the present invention. Platinumcompounds that are useful in treating cancer are known in the art andare described, for example in U.S. Pat. Nos. 4,994,591, 4,906,646,5,902,610, 5,053,226, 5,789,000, 5,871,710, 5,561,042, 5,604,095,5,849,790, 5,705,334, 4,863,902, 4,767,611, 5,670,621, 5,384,127,5,084,002, 4,937,262, 5,882,941, 5,879,917, 5,434,256, 5,393,909,5,117,022, 5,041,578, 5,843,475, 5,633,243, 5,178,876, 5,866,169,5,846,725, 5,646,011, 5,527,905, 5,844,001, 5,832,931, 5,676,978,5,604,112, 5,562,925, 5,541,232, 5,426,203, 5,288,887, 5,041,581,5,002,755, 4,946,954, 4,921,963, 4,895,936, 4,686,104, 4,594,238,4,581,224, 4,250,189, 5,829,448, 5,690,905, 5,665,771, 5,648,384,5,633,016, 5,460,785, 5,395,947, 5,256,653, 5,132,323, 5,130,308,5,106,974, 5,059,591, 5,026,694, 4,992,553, 4,956,459, 4,956,454,4,952,676, 4,895,935, 4,892,735, 4,843,161, 4,760,156, 4,739,087,4,720,504, 4,544,759, 4,515,954, 4,466,924, 4,462,998, 4,457,926,4,428,943, 4,325,950, 4,291,027, 4,291,023, 4,284,579, 4,271,085,4,234,500, 4,234,499, 4,200,583, 4,175,133, 4,169,846, 5,922,741,5,922,674, 5,922,302, 5,919,126, 5,910,102, 5,876,693, 5,871,923,5,866,617, 5,866,615, 5,866,593, 5,864,024, 5,861,139, 5,859,034,5,855,867, 5,855,748, 5,849,770, 5,843,993, 5,824,664, 5,821,453,5,811,119, 5,798,373, 5,786,354, 5,780,478, 5,780,477, 5,776,925,5,770,593, 5,770,222, 5,747,534, 5,739,144, 5,738,838, 5,736,156,5,736,119, 5,723,460, 5,697,902, 5,693,659, 5,688,773, 5,674,880,5,670,627, 5,665,343, 5,654,287, 5,648,362, 5,646,124, 5,641,627,5,635,218, 5,633,257, 5,632,982, 5,622,977, 5,622,686, 5,618,393,5,616,613, 5,612,019, 5,608,070, 5,595,878, 5,585,112, 5,580,888,5,580,575, 5,578,590, 5,575,749, 5,573,761, 5,571,153, 5,563,132,5,561,136, 5,556,609, 5,552,156, 5,547,982, 5,542,935, 5,525,338,5,519,155, 5,498,227, 5,491,147, 5,482,698, 5,469,854, 5,455,270,5,443,816, 5,415,869, 5,409,915, 5,409,893, 5,409,677, 5,399,694,5,399,363, 5,380,897, 5,340,565, 5,324,591, 5,318,962, 5,302,587,5,292,497, 5,272,056, 5,258,376, 5,238,955, 5,237,064, 5,213,788,5,204,107, 5,194,645, 5,182,368, 5,130,145, 5,116,831, 5,106,858,5,100,877, 5,087,712, 5,087,618, 5,078,137, 5,057,302, 5,049,396,5,034,552, 5,028,726, 5,011,846, 5,010,103, 4,985,416, 4,970,324,4,936,465, 4,931,553, 4,927,966, 4,912,072, 4,906,755, 4,897,384,4,880,832, 4,871,528, 4,822,892, 4,783,452, 4,767,874, 4,760,155,4,687,780, 4,671,958, 4,665,210, 4,645,661, 4,599,352, 4,594,418,4,593,034, 4,587,331, 4,575,550, 4,562,275, 4,550,169, 4,482,569,4,431,666, 4,419,351, 4,407,300, 4,394,319, 4,335,087, 4,329,299,4,322,391, 4,302,446, 4,287,187, 4,278,660, 4,273,755, 4,255,417,4,255,347, 4,248,840, 4,225,529, 4,207,416, 4,203,912, 4,177,263,4,151,185, 4,140,707, 4,137,248, 4,115,418, 4,079,121, 4,075,307,3,983,118, 3,870,719, RE 33,071, U.S. Pat. Nos. 6,087,392, 6,077,864,5,998,648, and 5,902,610, the disclosures of which are incorporated byreference herein in their entirety.

As is known in the art, platinum compounds are preferably used incombination with at least one epipodophyllotoxin compound, including,for example, etoposide (also known as VP-16) (available as VEPESID® fromBristol-Myers Squibb, Princeton, N.J.), teniposide (also known as VM-26)(available as VUMON® from Bristol-Myers Squibb, Princeton, N.J.), andthe like. Other epipodophyllotoxin compounds that become approved by theFDA or foreign counterparts thereof are also preferred for use in themethods and compositions of the present invention. Otherepipodophyllotoxin compounds that can be used in the present inventioninclude those described, for example, in U.S. Pat. Nos. 3,524,844,5,643,885, 5,066,645, 5,081,234, 5,891,724, 5,489,698, 5,821,348,5,571,914, 4,997,931, 4,547,567, 5,536,847, 5,326,753, 5,120,862,5,011,948, 4,895,727, 4,795,819, 4,644,072, 5,688,926, 5,676,978,5,660,827, 5,395,610, 5,346,897, 5,208,238, 5,190,949, 5,086,182,4,965,348, 4,958,010, 4,874,851, 4,866,189, 4,853,467, 4,728,740,4,716,221, 5,935,955, 5,863,538, 5,855,866, 5,776,427, 5,747,520,5,739,114, 5,622,960, 5,606,060, 5,605,826, 5,541,223, 5,459,248,5,455,161, 5,364,843, 5,300,500, 5,041,424, 5,036,055, 5,034,380,4,935,504, 4,916,217, 4,912,204, 4,904,768, 4,900,814, 4,888,419,4,567,253, RE 35,524, U.S. Pat. Nos. 6,107,284, 6,063,801, and6,051,230, the disclosures of which are incorporated herein by referencein their entirety.

Other compounds that can be used in the present invention includecamptothecin compounds. Camptothecin compounds are capable of inhibitingDNA topoisomerase I. Camptothecin compounds include camptothecin,derivatives of camptothecin and analogs of camptothecin. Camptothecincompounds are known in the art and include, for example, camptothecin,topotecan (available as HYCAMTIN® from SmithKline BeechamPharmaceuticals), CPT-11 (also called irinotecan), 9-aminocamptothecin,and the like. Other camptothecin compounds (or other compounds that caninhibit DNA topoisomerase I) that become approved by the FDA or foreigncounterparts thereof are also preferred for use in the methods andcompositions of the present invention. Other camptothecin compounds thatcan be used in the present invention include those described in, forexample, J. Med. Chem., 29:2358-2363 (1986); J. Med. Chem., 23:554(1980); J. Med. Chem., 30:1774 (1987); European Patent Application Nos.0 418 099, 0 088 642, and 0 074 770; and U.S. Pat. Nos. 5,633,016,5,004,758, 4,604,463, 4,473,692, 4,545,880, 4,513,138, 4,399,276,6,121,451, 6,121,278, 6,121,277, 6,121,275, 6,121,263, 6,107,486,6,100,273, 6,096,336, 6,093,721, 6,063,801, 6,046,209, 6,040,313,6,034,243, 6,028,078, 5,998,426, 5,990,120, 5,985,888, 5,981,542,5,972,955, 5,968,943, 5,958,937, 5,955,467, 5,948,797, 5,935,967,5,932,709, 5,932,588, 5,922,877, 5,916,897, 5,916,896, 5,910,491,5,900,419, 5,892,043, 5,889,017, 5,880,133, 5,859,023, 5,859,022,5,856,333, 5,843,954, 5,840,899, 5,837,673, 5,834,012, 5,807,874,5,801,167, 5,786,344, 5,773,522, 5,767,142, 5,744,605, 5,734,056,5,731,316, 5,726,181, 5,677,286, 5,674,874, 5,674,873, 5,670,500,5,633,177, 5,652,244, 5,646,159, 5,633,260, 5,614,628, 5,604,233,5,602,141, 5,597,829, 5,559,235, 5,552,154, 5,541,327, 5,525,731,5,496,952, 5,475,108, 5,468,859, 5,468,754, 5,459,269, 5,447,936,5,446,047, 5,401,747, 5,391,745, 5,364,858, 5,340,817, 5,244,903,5,227,380, 5,200,524, 5,191,082, 5,180,722, 5,162,532, 5,122,606,5,122,526, 5,106,742, 5,061,800, 5,053,512, 5,049,668, 5,004,758,4,981,968, 4,943,579, 4,939,255, 4,914,205, 4,894,456, RE 32,518, U.S.Pat. Nos. 4,604,463, 4,513,138, 4,473,692, 4,399,282, 4,399,276, and4,031,098, the disclosures of which are incorporated by reference hereinin their entirety.

The immunoconjugates and chemotherapeutic agents of the presentinvention can be administered in vitro, in vivo and/or ex vivo to treatpatients and/or to modulate the growth of selected cell populationsincluding, for example, cancer of the lung, breast, colon, prostate,kidney, pancreas, brain, bones, ovary, testes, and lymphatic organs;autoimmune diseases, such as systemic lupus, rheumatoid arthritis, andmultiple sclerosis; graft rejections, such as renal transplantrejection, liver transplant rejection, lung transplant rejection,cardiac transplant rejection, and bone marrow transplant rejection;graft versus host disease; viral infections, such as CMV infection, HIVinfection, and AIDS; and parasite infections, such as giardiasis,amoebiasis, schistosomiasis, and the like. Preferably, theimmunoconjugates and chemotherapeutic agents of the invention areadministered in vitro, in vivo and/or ex vivo to treat cancer in apatient and/or to modulate the growth of cancer cells, including, forexample, cancer of the lung, breast, colon, prostate, kidney, pancreas,brain, bones, ovary, testes, and lymphatic organs; more preferably lungcancer or colon cancer. In a most preferred embodiment, the lung canceris small cell lung cancer (SCLC).

“Modulating the growth of selected cell populations” includes inhibitingthe proliferation of selected cell populations (e.g., SCLC cells, NCIN417 cells, SW-2 cells, NCI-H441 cells, HT-29 cells, and the like) fromdividing to produce more cells; reducing the rate of increase in celldivision as compared, for example, to untreated cells; killing selectedcell populations; and/or preventing selected cell populations (such ascancer cells) from metastasizing. The growth of selected cellpopulations can be modulated in vitro, in vivo or ex vivo.

In the methods of the present invention, the immunoconjugates andchemotherapeutic agents can be administered in vitro, in vivo, or exvivo separately or as components of the same composition. Theimmunoconjugates and chemotherapeutic agents can be used with suitablepharmaceutically acceptable carriers, diluents, and/or excipients, whichare well known, and can be determined by one of skill in the art as theclinical situation warrants. Examples of suitable carriers, diluentsand/or excipients include: (1) Dulbecco's phosphate buffered saline, pHabout 6.5, which would contain about 1 mg/ml to 25 mg/ml human serumalbumin, (2) 0.9% saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose.

The compounds and compositions described herein may be administered inappropriate form, preferably parenterally, more preferablyintravenously. For parenteral administration, the compounds orcompositions can be aqueous or nonaqueous sterile solutions, suspensionsor emulsions. Propylene glycol, vegetable oils and injectable organicesters, such as ethyl oleate, can be used as the solvent or vehicle. Thecompositions can also contain adjuvants, emulsifiers or dispersants. Thecompositions can also be in the form of sterile solid compositions whichcan be dissolved or dispersed in sterile water or any other injectablesterile medium.

The “therapeutically effective amount” of the chemotherapeutic agentsand immunoconjugates described herein refers to the dosage regimen forinhibiting the proliferation of selected cell populations and/ortreating a patient's disease, and is selected in accordance with avariety of factors, including the age, weight, sex, diet and medicalcondition of the patient, the severity of the disease, the route ofadministration, and pharmacological considerations, such as theactivity, efficacy, pharmacokinetic and toxicology profiles of theparticular compound used. The “therapeutically effective amount” canalso be determined by reference to standard medical texts, such as thePhysicians Desk Reference 1999 (53rd Ed.), the disclosure of which isincorporated by reference herein in its entirety. The patient ispreferably an animal, more preferably a mammal, most preferably a human.The patient can be male or female, and can be an infant, child or adult.

Examples of suitable protocols of immunoconjugate administration are asfollows. Immunoconjugates can be given daily for about 5 days either asan i.v. bolus each day for about 5 days, or as a continuous infusion forabout 5 days. Alternatively, they can be administered once a week forsix weeks or longer. As another alternative, they can be administeredonce every two or three weeks. Bolus doses are given in about 50 toabout 400 ml of normal saline to which about 5 to about 10 ml of humanserum albumin can be added. Continuous infusions are given in about 250to about 500 ml of normal saline, to which about 25 to about 50 ml ofhuman serum albumin can be added, per 24 hour period. Dosages will beabout 10 μg to about 1000 mg/kg per person, i.v. (range of about 100 ngto about 10 mg/kg). About one to about four weeks after treatment, thepatient can receive a second course of treatment. Specific clinicalprotocols with regard to route of administration, excipients, diluents,dosages, and times can be determined by the skilled artisan as theclinical situation warrants.

The present invention also provides pharmaceutical kits comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compounds and/or compositions of the present invention,including, one or more immunoconjugates and one or more chemotherapeuticagents. Such kits can also include, for example, other compounds and/orcompositions, a device(s) for administering the compounds and/orcompositions, and written instructions in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products.

EXAMPLES

The following examples are for purposes of illustration only, and arenot intended to limit the scope of the invention or claims.

Example 1

The maytansinoid DM1 was conjugated to the humanized monoclonal antibodyN901.

Ansamitocin P-3, provided by Takeda (Osaka, Japan) was converted to thedisulfide-containing maytansinoid DM1, as described herein and in U.S.Pat. No. 5,208,020, the disclosure of which is incorporated by referenceherein in its entirety.

Humanized N901 is an antibody that binds to the CD56 antigen expressedon all human small cell lung cancer (SCLC) tissues, neuroblastomas andcarcinoid tumors (Doria et al, Cancer 62:1839-1845 (1988); Kibbelaar etal, Eur. J. Cancer, 27:431-435 (1991);Rygaard et al, Br. J. Cancer,65:573-577 (1992)).

Humanized N901 was modified withN-succinimidyl-4-[2-pyridyldithio]-pentanoate (SPP) to introducedithiopyridyl groups. Alternatively,N-succinimidyl-3-[2-pyridyldithio]-propanoate (SPDP) can be used. Theantibody (8 mg/mL) in 50 mM potassium phosphate buffer, pH 6.5,containing NaCl (50 mM) and EDTA (2 mM) was treated with SPP (5.6 molarequivalents in ethanol). The final ethanol concentration was 5% (v/v).After 90 minutes at ambient temperature, the reaction mixture was gelfiltered through a Sephadex G25 column equilibrated in the above buffer.Antibody-containing fractions were pooled and the degree of modificationwas determined by treating a sample with dithiothreitol and measuringthe change in absorbance at 343 nm (release of 2-mercaptopyridine withε_(343 nm)=8,080 M⁻¹). Recovery of the antibody was typically 90%, with4.8 pyridyldithio groups linked per antibody molecule.

The modified antibody was diluted with 50 mM potassium phosphate buffer,6.5, containing NaCl (50 mM) and EDTA (2 mM) to a final concentration of2.5 mg/mL. DM1 (1.7 eq.) in dimethylacetamide (DMA, 3% v/v in finalreaction mixture) was then added to the modified antibody solution. Thereaction proceeded at ambient temperature under argon for 20 hours.

The reaction mixture was then loaded on to a Sephacryl S300 gelfiltration column equilibrated with phosphate-buffered saline (PBS, pH6.5). The major peak comprised monomeric hu901-DM1. The number of DM1drug molecules linked per antibody molecule was determined by measuringthe absorbance at 252 nm and 280 nm and found to be 3.9 DM1 moleculesper antibody molecule.

Example 2

In this experiment, a low, non-curative dose of huN901-DM1 was used withan optimal does of paclitaxel (Sigma Chemical Co., St. Louis, Mo.). SCIDmice (7 animals per group) were inoculated subcutaneously with NCI N417cells (8×10⁶ cells/animal). After the tumors were well-established(average tumor size was approximately 100 mm³), one group of animals wastreated with huN901-DM1 at a DM1 dose of 75 μg/kg/d×5, administered i.v.everyday. A second group of animals was treated with paclitaxel at adose of 10 mg/kg/d×5, administered by i.p. everyday. A third group ofanimals was treated with huN901-DM1 and paclitaxel using the same doseand schedule used for the individual agents. A fourth, control group ofanimals was left untreated. Tumor size was measured as described by Liuet al, Proc. Natl. Acad. Sci., 93:8618-8623 (1996). Animals were alsomonitored for weight loss as an indicator of signs of toxicity.

The results of the experiment are shown in FIG. 5. In the control groupof animals, the tumors grew rapidly to a size of about 900 mm³ by Day 28post-tumor inoculation. In animals treated with either huN901-DM1 orpaclitaxel, there was a modest anti-tumor effect with a tumor growthdelay of 4 days in each case. In the animals treated with huN901-DM1 andpaclitaxel, the tumors disappeared with complete regression lasting 58days. Importantly, there was no evidence of toxicity in the animals.These data demonstrate that treatment with huN901-DM1 and paclitaxel hasan unexpectedly superior (e.g., synergistic) anti-tumor effect.

Example 3

In this experiment, a low, non-curative dose of huN901-DM1 was used withan optimal dose of cisplatin (Sigma Chemical Co., St. Louis, Mo.) andetoposide (Sigma Chemical Co., St. Louis, Mo.). SCID mice (7 animals pergroup) were inoculated subcutaneously with NCI N417 cells (8×10⁶cells/animal). After the tumors were well-established (average tumorsize was approximately 100 mm³), one group of animals was treated withhuN901-DM1 at a DM1 dose of 75 μg/kg/d×5, administered i.v. everyday. Asecond group of animals was treated with cisplatin (at a dose of 2mg/kg/d×3, administered by i.v. ever other day) and etoposide (at a doseof 8 mg/kg/d×3, administered every other day). A third group of animalswas treated with huN901-DM1, cisplatin and etoposide using the same doseand schedule used for a the individual agents. A fourth, control groupof animals was left untreated. Tumor size was measured as described byLiu et al, Proc. Natl. Acad. Sci., 93:8618-8623 (1996). Animals werealso monitored for weight loss as an indicator of signs of toxicity.

The results of the experiment are shown in FIG. 6. In the control groupof animals, the tumors grew rapidly to a size of about 900 mm³ by Day 28post-tumor inoculation. In animals treated with either huN901-DM1 orcisplatin and etoposide, there was a modest anti-tumor effect with atumor growth delay of 4 days in each case. In the animals treated withhuN901-DM1, cisplatin and etoposide, there was a tumor growth delay of12 days, which is 50% longer than what one would expect for an additiveanti-tumor effect of the individual compounds. Importantly, there was noevidence of toxicity in the animals. These data demonstrate thattreatment with huN901-DM1, cisplatin, and etoposide has an unexpectedlysuperior (e.g., synergistic) anti-tumor effect.

Example 4

The anti-tumor effect of a combination of a low dose of huN901-DM1 anddocetaxel (available as TAXOTERE® from Aventis) was evaluated in anestablished subcutaneous xenograft model of small cell lung cancer. SCIDmice (24 animals) were inoculated with human small cell lung cancer SW-2cells (8×10⁶ cells/animal) injected subcutaneously into the right flankof the mice. When the tumors reached about 100 mm³ in size (10 daysafter tumor cell inoculation), the mice were randomly divided into fourgroups (6 animals per group). The first group of mice was treated withdocetaxel (5 mg/kg×5, q2d) administered i.v. A second group of animalswas treated with huN901-DM1 (DM1 dose of 75 μg/kg×5, qd) administeredi.v. The third group of mice received a combination of docetaxel andhuN901-DM1, using the same doses and schedules as in groups 1 and 2. Acontrol group of animals received phosphate-buffered saline (PBS) usingthe same schedule as the animals in group 2. Tumor growth was monitoredby measuring tumor size twice per week. Tumor size was calculated withthe formula: length×width×height×½.

The change in tumor size is shown in FIG. 7. In the control group ofanimals, tumors grew rapidly to about 1000 mm³ in 26 days. Treatmentwith docetaxel alone, or a low dose of huN901-DM1 alone, resulted intumor growth delays of 8 days and 20 days, respectively. In contrast,treatment with the combination of docetaxel and huN901-DM1 showed aremarkable anti-tumor effect resulting in complete tumor regression inall the treated animals. In 3 out of 6 animals in this treatment group,the tumor was eradicated—resulting in cures lasting greater than 200days. In the remaining 3 animals in this group, there was a tumor growthdelay of 52 days, which is 24 days longer than the calculated additiveeffect. Thus, the combination of docetaxel and huN901-DM1 shows anunexpectedly superior (e.g., synergistic) anti-tumor effect in thishuman SCLC xenograft model.

Example 5

The anti-tumor effect of a combination of a low dose of huN901-DM1 andtopotecan (available as HYCAMTIN® from SmithKline BeechamPharmaceuticals), one of the approved drugs for the treatment of smallcell lung cancer (SCLC) in humans, was evaluated in an establishedsubcutaneous xenograft model of SCLC. SCID mice (24 animals) wereinoculated with human small cell lung cancer SW-2 cells (8×10⁶cells/animal) injected subcutaneously into the right flank of the mice.When the tumors reached about 80 mm³ in size, the mice were randomlydivided into four groups (6 animals per group). The first group of micewas treated with topotecan (1.4 mg/kg×5, qd) administered i.v. A secondgroup of animals was treated with huN901-DM1 (DM1 dose of 100 μg/kg×5,qd) administered i.v. The third group of mice received a combination oftopotecan and huN901-DM1, using the same doses and schedules as ingroups 1 and 2. A control group of animals received phosphate-bufferedsaline (PBS) using the same schedule as the animals in group 2. Tumorgrowth was monitored by measuring tumor size twice per week. Tumor sizewas calculated using the formula: length×width×height×½.

The change in tumor size is shown in FIG. 8. In the control group ofanimals, tumors grew to about 800 mm³ in 44 days. Treatment withtopotecan alone resulted in tumor growth delays of 12 days. Treatmentwith a low dose of huN901-DM1 alone resulted in a tumor-growth delay of34 days in 3 out of 6 animals. The remaining 3 animals in this group hadcomplete tumor regressions. Treatment with the combination of topotecanand huN901-DM1 showed a remarkable anti-tumor effect resulting incomplete tumor regression in 5 out of the 6 treated animals. Theseanimals were tumor-free on day 78, the last measurement point. Thus, thecombination of topotecan and huN901-DM1 is unexpectedly superior (e.g.,synergistic) when compared to the single agents in this human SCLCxenograft model.

Example 6

The anti-tumor effect of a combination of a low dose of huC242-DM1(manufactured by ImmunoGen, Inc. following the procedures described inU.S. Pat. No. 5,208,020, the disclosure of which is incorporated byreference herein in its entirety, and also described in Example 1) andpaclitaxel (Sigma Chemical Co., St. Louis, Mo.) was evaluated in anestablished subcutaneous xenograft model of non-small cell lung cancer.SCID mice (24 animals) were inoculated with human lung adenocarcinomaNCI-H441 cells (8×10⁶ cells/animal), injected subcutaneously into theright flank of the mice. When the tumors reached about 125 mm³ in size(4 days after tumor cell inoculation), the mice were randomly dividedinto four groups (6 animals per group). The first group of mice wastreated with paclitaxel (15 mg/kg×5, q2d) administered i.p. A secondgroup of animals was treated with huC242-DM1 (DM1 dose of 75 μg/kg×5,qd) administered i.v. The third group of mice received a combination ofpaclitaxel and huC242-DM1, using the same doses and schedules as ingroups 1 and 2. In the combination group, the huC242-DM1 conjugate wasadministered 2 hours after the paclitaxel. A control group of animalsreceived phosphate-buffered saline (PBS) using the same schedule as theanimals in group 2. Tumor growth was monitored by measuring tumor sizetwice per week. Tumor size was calculated using the formula:length×width×height×½.

The change in tumor size is shown in FIG. 9. In the control group ofanimals, tumors grew rapidly to about 1000 mm³ in 32 days. Treatmentwith paclitaxel alone, resulted in a small tumor growth delay of 4 days.Treatment with huC242-DM1 resulted in shrinkage of the tumor, but noneof the 6 treated animals showed complete tumor regression. Treatmentwith a combination of paclitaxel and huC242-DM1 showed a greateranti-tumor effect resulting in complete tumor regression, with 3 out ofthe 6 animals showing no evidence of tumor. The remaining 3 animals inthis group showed a significant shrinkage in the tumor. Thus, thecombination of paclitaxel and huC242-DM1 is unexpectedly superior (e.g.,synergistic) in this human SCLC lung adenocarcinoma xenograft model.

Example 7

The anti-tumor effect of a combination of a low dose of huC242-DM1(manufactured by ImmunoGen, Inc. following the methods described in U.S.Pat. No. 5,208,020, the disclosure of which is incorporated by referenceherein in its entirety, and also described in Example 1) and paclitaxel(Sigma Chemical Co., St. Louis, Mo.) was evaluated in an establishedsubcutaneous xenograft model of non-small cell lung cancer. SCID mice(32 animals) were inoculated with human colon cancer HT-29 cells (8×10⁶cells/animal), injected subcutaneously into the right flank of the mice.When the tumors reached about 80 mm³ in size, the mice were randomlydivided into four groups (8 animals per group). The first group of micewas treated with CPT-11 (50 mg/kg×2, q3d) administered i.v. The secondgroup of animals was treated with murine C242-DM1 (DM1 dose of 75μg/kg×5, qd) administered i.v. The third group of mice received acombination of CPT-11 and C242-DM1, using the same doses and schedulesas in groups 1 and 2. A control group of animals receivedphosphate-buffered saline (PBS) using the same schedule as the animalsin group 2. Tumor growth was monitored by measuring tumor size twice perweek. Tumor size was calculated using the formula:length×width×height×½.

The change in tumor size is shown in FIG. 10. In the control group ofanimals, tumors grew rapidly to about 1000 mm³ in 31 days. Treatmentwith CPT-11 alone resulted in a small tumor growth delay of 6 days.Treatment with C242-DM1 resulted in a delay in tumor growth of 22 days.Treatment with a combination of CPT-11 and C242-DM1 showed anunexpectedly superior anti-tumor effect resulting in a tumor growthdelay of 38 days, which is 10 days longer than the calculated additiveeffect. Thus, the combination of CPT-11 and C242-DM1 is unexpectedlysuperior (e.g., synergistic) in this human colon cancer xenograft model.

Each of the patents and publications cited in the specification isincorporated by reference herein in its entirety.

Various modifications of the invention, in addition to those describedherein, will be apparent to one skilled in the art from the foregoingdescription. Such modifications are intended to fall within the scope ofthe appended claims.

1. A pharmaceutical composition comprising a synergistic combination ofat least one chemotherapeutic agent and at least one immunoconjugate;wherein the immunoconjugate comprises at least one maytansinoid compoundlinked to a monoclonal antibody or fragment thereof; wherein themonoclonal antibody or fragment thereof binds to an antigen expressed bya cancer cell, and wherein the chemotherapeutic agent is a taxanecompound, an epothilone compound, a platinum compound, anepipodophyllotoxin compound, a camptothecin compound, or a mixture oftwo or more thereof.
 2. The pharmaceutical composition of claim 1,wherein the chemotherapeutic agent is a taxane compound, a platinumcompound, an epipodophyllotoxin compound, a camptothecin compound, or amixture of two or more thereof.
 3. The pharmaceutical composition ofclaim 1, wherein the chemotherapeutic agent is paclitaxel, docetaxel,epothilone A, epothilone B, epothilone C, epothilone D, epothilone E,epothilone F, cisplatin, carboplatin, oxaliplatin, iproplatin,ormaplatin, tetraplatin, etoposide, teniposide, camptothecin, topotecan,irinotecan, 9-aminocamptothecin, or a mixture of two or more thereof. 4.The pharmaceutical composition of claim 1, wherein the chemotherapeuticagent is paclitaxel, cisplatin, etoposide, docetaxel, topotecan, or amixture of two or more thereof.
 5. The pharmaceutical composition ofclaim 1, wherein the monoclonal antibody or fragment thereof binds to aCD56 antigen.
 6. The pharmaceutical composition of claim 1, wherein themonoclonal antibody or fragment thereof is at least one of Fv, Fab, Fab′or F(ab′)₂.
 7. The pharmaceutical composition of claim 1, wherein themonoclonal antibody or fragment thereof is humanized N901.
 8. Thepharmaceutical composition of claim 1, wherein the monoclonal antibodyor fragment thereof is humanized C242.
 9. The pharmaceutical compositionof claim 1, wherein the immunoconjugate comprises at least onemaytansinoid compound of formula (IV):

wherein is Z₀ is H or SR; R is methyl, linear alkyl, branched alkyl,cyclic alkyl, simple or substituted aryl or heterocyclic; t is 1, 2 or3; Y₀ is chlorine or hydrogen; and X₃ is hydrogen or methyl.
 10. Thepharmaceutical composition of claim 9, wherein Z₀ is H; t is 2; Y₀ ischlorine; and X₃ is methyl.
 11. The pharmaceutical composition of claim1, wherein the immunoconjugate is of the formula:

wherein MAb is a monoclonal antibody or fragment thereof that binds toan antigen expressed by the cancer cell.
 12. A pharmaceuticalcomposition comprising a synergistic combination of at least onechemotherapeutic agent and at least one immunoconjugate; wherein thechemotherapeutic agent is a taxane compound, an epothilone compound, aplatinum compound, an epipodophyllotoxin compound, a camptothecincompound, or a mixture of two or more thereof; and wherein theimmunoconjugate is:

wherein MAb is a monoclonal antibody or fragment thereof that binds toan antigen expressed by a cancer cell.
 13. A pharmaceutical compositioncomprising a synergistic combination of (i) at least onechemotherapeutic agent selected from the group consisting of paclitaxel,docetaxel, cisplatin, etoposide, topotecan and irinotecan and (ii) animmunoconjugate comprising a maytansinoid and a humanized monoclonalantibody selected from the group consisting of N901 and C242.
 14. Thepharmaceutical composition of claim 13, wherein the maytansinoid is acompound of formula (IV):

wherein Z₀ is H or SR; wherein R is methyl, linear alkyl, branchedalkyl, cyclic alkyl, simple or substituted aryl or heterocyclic; t is 1,2 or 3; Y₀ is chlorine or hydrogen; and X₃ is hydrogen or methyl.
 15. Apharmaceutical composition comprising a synergistic combination of (i)at least one chemotherapeutic agent selected from the group consistingof paclitaxel, docetaxel, cisplatin, etoposide, topotecan and irinotecanand (ii) an immunoconjugate comprising a maytansinoid and a humanizedmonoclonal antibody or fragment thereof that binds to an antigenexpressed by a small cell lung cancer cell, a non small cell lung cancercell or a colorectal cancer cell.
 16. The pharmaceutical composition ofclaim 15, wherein the maytansinoid is a compound of formula (IV):

wherein Z₀ is H or SR; wherein R is methyl, linear alkyl, branchedalkyl, cyclic alkyl, simple or substituted aryl or heterocyclic; t is 1,2 or 3; Y₀ is chlorine or hydrogen; and X₃ is hydrogen or methyl.
 17. Animmunoconjugate comprising a cell binding agent and a maytansinoid,wherein the cell binding agent is conjugated to the maytansinoid usingN-succinimidyl-4-[2-pyridyldithio]-pentanoate (SPP), and wherein thecell binding agent is a humanized N901 monoclonal antibody or an antigenbinding fragment thereof.
 18. The immunoconjugate of claim 17, whereinthe cell binding agent is a humanized N901 monoclonal antibody.
 19. Theimmunoconjugate of claim 17, wherein the cell binding agent is an Fv,Fab, Fab′, or F(ab′)₂ fragment of a humanized N901 monoclonal antibody.20. The immunoconjugate of claim 17, wherein the maytansinoid is DM1.21. A pharmaceutical composition comprising the immunoconjugate of claim17 and a pharmaceutically acceptable carrier.